Structure Activity Relationship of Carbonic Anhydrase Inhibitors

Structure Activity Relationship of Carbonic Anhydrase Inhibitors using Surface Plasmon Resonance:

Structure Activity Relationship of Carbonic Anhydrase Inhibitors using Surface Plasmon Resonance By Nat Dempkowski

Materials:

Materials Biacore T100 Biacore CM5 Sensor Chip Human Carbonic Anhydrase I Bovine Carbonic Anhydrase II Binding and Buffer Solutions EA, EDC, NHS, Acetate, PBS, HEPES, NaOH , Sulfamate Carbonic Anhydrase Inhibitors (Sulfonamides) Human Carbonic Anhydrase II

Inhibitors:

Inhibitors Sulfanilamide Acetazolamide Celecoxib Ammonium Sulfamate Topiramate Sulpiride Hydrochlorothiazide Methazolamide Saccharin

Protein Immobilization:

Protein Immobilization Procedure Biacore T100 Prime CM5 sensor chip by running NaOH and Sulfamate into the flow cells Using EA, EDC, NHS solutions, immobilize Carbonic Anhydrase on the chip Measure Response Units of Carbonic Anhydrase immobilized on chip CM5 Sensor Chip

Inhibitor Response Measurement:

Inhibitor Response Measurement Procedure Dilute inhibitors in buffer solution Dilute different concentrations of inhibitors from ~500µM to ~1µM Inject solutions into flow cells with immobilized protein from lowest concentration to highest Run buffer injections after high concentrations to clear accumulation of inhibitors Measure response units using Surface P lasmon R esonance and model kinetics of binding

Response from Inhibitors:

Response from Inhibitors Celecoxib Hydrochlorothiazide Methazolamide Sulfanilamide Topiramate Acetazolamide

Kinetics Data:

Kinetics Data Mw (g/mol) Ka (association) Kd (dissociation) KD (affinity) Rmax (RU) Acetazolamide 222.3 5.42E+06 0.05723 1.06E-08 16.82 Celecoxib 381.4 8.13E+04 0.1267 1.56E-06 26.31 Hydrochlorothiazide 297.7 1.16E+04 0.03752 3.25E-06 22.46 Methazolamide 236.3 5.79E+06 0.07977 1.38E-08 19.51 Sulfanilamide 172.2 2.40E+04 0.05141 2.14E-06 14.99 Topiramate 339.4 8.75E+04 0.02324 2.66E-07 13.32 Ammonium Sulfamate 114.1 No Binding Observed Sodium saccharin 205.2 No Binding Observed Sulpiride 341.4 No Binding Observed

Association Rates:

Association Rates

Binding Affinity:

Binding Affinity

Conclusion:

Conclusion Binding Affinity is determined largely by the Stiffness/Flatness of Molecule Double bonded nitrogen group increases association rates greatly. Acetazolamide Methazolamide